Polarity of the cytoskeleton is essential for many cell behaviors, including directed migration during wound healing and chemotaxis. Microtubule polymerization dynamics are spatiotemporally regulated in planar polarized, migrating cells, and are required for cells to migrate directionally. The overall objective of this proposal is to determine the mechanisms by which microtubules function during establishment and maintenance of planar polarity and directed sheet migration of epithelial cells with an emphasis on the function and dynamics of the CLASP family of +TIPs. +TIPs are a heterogeneous group of proteins defined by their dynamic localization to growing microtubule plus ends in cells. +TIPs are prime candidates to mediate microtubule function during epithelial sheet migration because they may provide interfaces for regulated interactions of microtubule ends with the cortical cytoskeleton. The current study focuses on CLASPs because they are distinguished from other +TIPs by their spatiotemporally regulated association with microtubules in migrating epithelial cells. Although CLASPs track microtubule plus ends in the cell body, in contrast to other +TIPs CLASPs associate along microtubules in the lamella of migrating epithelial cells. This association of CLASPs along lamella microtubules is decreased through phosphorylation by glycogen synthase kinase 3? (GSK3?), an emerging integrator of cell polarity pathways. Because GSK3? is thought to be locally inactivated in the front of migrating cells, it is predicted that CLASP-phosphorylation in the cell body decreases CLASP-microtubule affinity, while association of non-phosphorylated CLASPs with lamella microtubules stabilizes these microtubules. The specific hypothesis of this proposal is that CLASP- mediated lamella microtubule stabilization and interactions of these microtubules with the cortical cytoskeleton are essential for persistent planar polarity of migrating epithelial cells. This hypothesis will be tested by biochemical methods and advanced live cell microscopy in clonal human keratinocyte cells. In Aim 1, GSK3?-dependent CLASP phosphorylation will be analyzed in cells and in vitro, and it will be tested whether CLASP interactions with microtubules or associated proteins are regulated by phosphorylation. In Aim 2, CLASP-mediated spatiotemporal regulation of microtubule polymerization dynamics will be analyzed in planar polarized, migrating cells, and in vitro with purified components. It will be tested whether CLASPs directly or indirectly regulate microtubule dynamics and whether this is regulated by GSK3? phosphorylation. In Aim 3, CLASP function will be inhibited in migrating epithelial cells by RNA interference, and it will be determined how CLASPs regulate directed cell migration by analyzing lamella protrusion, cell- matrix adhesion, and polarity dynamics using live cell spinning disk confocal microscopy. PUBLIC HEALTH RELEVANCE: Collective cell migration is essential for many morphogenetic movements and the sheet migration of keratinocytes across the basement membrane after injury. In addition, abnormal regulation of cell migration contributes to metastasis of tumor cells, and collective migration of tumor cells plays a crucial role in tumor invasiveness. Understanding the molecular mechanisms underlying planar epithelial cell polarity and directed migration is thus crucial to developing improved therapies for keratinocyte or other carcinomas, and for skin regeneration after wounding.